*Target (protein/gene name): Phosphotyrosine protein phosphatase (ptpB) *NCBI Gene # or RefSeq#: 886842 *Protein ID (NP or XP #) or Wolbachia#: NP_214667 *Organism (including strain): Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) Etiologic Risk Group (see link below): Risk Group 3 *Disease Information (sort of like the Intro to your Mini Research Write up):
M. tuberculosis causes tuberculosis, a potentially fatal infectious pulmonary disease. Tuberculosis can be spread through the air through small droplets containing the bacteria from the coughs of sneezes of infected persons. Tuberculosis has become more common in developed countries due to the spread of HIV, which makes patients more susceptible to tuberculosis. Tuberculosis occurs in both a latent and active form and results in symptoms including coughing up blood, chest pain, weight loss, fatigue, fever, chills, and loss of appetite. Especially common in low and middle income countries, tuberculosis has the second highest worldwide mortality count for a single infectious agent. Treatment for tuberculosis currently requires multiple drugs for at least six months and without proper treatment, up to 66% of patients will die. Link to TDR Targets page (if present): Not present Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://www.ncbi.nlm.nih.gov/gene/886842 Essentiality of this protein: “essential virulence factor that is secreted by the bacterium into the cytoplasm of macrophages, where it mediates mycobacterial survival in the host” (https://www.ncbi.nlm.nih.gov/pubmed/20167798) Is it a monomer or multimer as biological unit?: Monomer Complex of proteins?: One of three phosphatases (PtpA, PtpB, and SapM) Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Homo sapiens –“MptpB is a promising target for development of anti-tuberculosis drugs” - http://www.brenda-enzymes.org/literature.php?e=3.1.3.48&r=710432 Macaca fascicularis - “PTP-1B antagonizes insulin signaling and is a potential therapeutic target for insulin resistance associated with obesity and type 2 diabetes” - http://www.brenda-enzymes.org/literature.php?e=3.1.3.48&r=692092
Current Inhibitors: I-A09 (https://www.ncbi.nlm.nih.gov/pubmed/20167798) Expression Information (has it been expressed in bacterial cells): Expressed in e.Coli Purification Method: immobilized metal affinity chromatography, and size exclusion chromatography (can also use a cobalt charged resin column) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775457/ http://www.biomedcentral.com/1471-2180/13/292 Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MAVRELPGAWNFRDVADTATALRPGRLFRSSELSRLDDAGRATLRRLGITDVADLRSSREVARRGPGRVP
DGIDVHLLPFPDLADDDADDSAPHETAFKRLLTNDGSNGESGESSQSINDAATRYMTDEYRQFPTRNGAQ
RALHRVVTLLAAGRPVLTHCFAGKDRTGFVVALVLEAVGLDRDVIVADYLRSNDSVPQLRARISEMIQQR
FDTELAPEVVTFTKARLSDGVLGVRAEYLAAARQTIDETYGSLGGYLRDAGISQATVNRMRGVLLG *length of your protein in Amino Acids: 276aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
30.1538 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 14440 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
atggcggtgcgcgaactgccgggcgcgtggaactttcgcgatgtggcggataccgcgacc gcgctgcgcccgggccgcctgtttcgcagcagcgaactgagccgcctggatgatgcgggc cgcgcgaccctgcgccgcctgggcattaccgatgtggcggatctgcgcagcagccgcgaa gtggcgcgccgcggcccgggccgcgtgccggatggcattgatgtgcatctgctgccgttt ccggatctggcggatgatgatgcggatgatagcgcgccgcatgaaaccgcgtttaaacgc ctgctgaccaacgatggcagcaacggcgaaagcggcgaaagcagccagagcattaacgat gcggcgacccgctatatgaccgatgaatatcgccagtttccgacccgcaacggcgcgcag cgcgcgctgcatcgcgtggtgaccctgctggcggcgggccgcccggtgctgacccattgc tttgcgggcaaagatcgcaccggctttgtggtggcgctggtgctggaagcggtgggcctg gatcgcgatgtgattgtggcggattatctgcgcagcaacgatagcgtgccgcagctgcgc gcgcgcattagcgaaatgattcagcagcgctttgataccgaactggcgccggaagtggtg acctttaccaaagcgcgcctgagcgatggcgtgctgggcgtgcgcgcggaatatctggcg gcggcgcgccagaccattgatgaaacctatggcagcctgggcggctatctgcgcgatgcg ggcattagccaggcgaccgtgaaccgcatgcgcggcgtgctgctgggc *GC% Content for gene: 65.821% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#: 886842
*Protein ID (NP or XP #) or Wolbachia#: NP_214667
*Organism (including strain): Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Etiologic Risk Group (see link below): Risk Group 3
*Disease Information (sort of like the Intro to your Mini Research Write up):
M. tuberculosis causes tuberculosis, a potentially fatal infectious pulmonary disease. Tuberculosis can be spread through the air through small droplets containing the bacteria from the coughs of sneezes of infected persons. Tuberculosis has become more common in developed countries due to the spread of HIV, which makes patients more susceptible to tuberculosis. Tuberculosis occurs in both a latent and active form and results in symptoms including coughing up blood, chest pain, weight loss, fatigue, fever, chills, and loss of appetite. Especially common in low and middle income countries, tuberculosis has the second highest worldwide mortality count for a single infectious agent. Treatment for tuberculosis currently requires multiple drugs for at least six months and without proper treatment, up to 66% of patients will die.
Link to TDR Targets page (if present): Not present
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://www.ncbi.nlm.nih.gov/gene/886842
Essentiality of this protein: “essential virulence factor that is secreted by the bacterium into the cytoplasm of macrophages, where it mediates mycobacterial survival in the host” (https://www.ncbi.nlm.nih.gov/pubmed/20167798)
Is it a monomer or multimer as biological unit?: Monomer
Complex of proteins?: One of three phosphatases (PtpA, PtpB, and SapM)
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Homo sapiens –“MptpB is a promising target for development of anti-tuberculosis drugs” - http://www.brenda-enzymes.org/literature.php?e=3.1.3.48&r=710432
Macaca fascicularis - “PTP-1B antagonizes insulin signaling and is a potential therapeutic target for insulin resistance associated with obesity and type 2 diabetes” - http://www.brenda-enzymes.org/literature.php?e=3.1.3.48&r=692092
*EC#: 3.1.3.48
Link to BRENDA EC# page: http://www.brenda-enzymes.info/enzyme.php?ecno=3.1.3.48
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): continuous spectrophotometric assay
-- link to Sigma (or other company) page for assay (see Sigma links below)
https://www.neb.com/products/p0757-p-nitrophenyl-phosphate-pnpp
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097125/
-- links to assay reagents (substrates) pages; List cost and quantity of substrate reagents, supplier, and catalog #
-NaOH (Sigma Aldrich, #221465, $36.00 for 25g)
http://www.sigmaaldrich.com/catalog/product/sial/221465?lang=en®ion=US
-pNPP (Sigma Aldrich, #N7653, $89.60 per pack)
http://www.sigmaaldrich.com/catalog/product/sigma/n7653?lang=en®ion=US
-Alkaline Phosphatase Stabilizing Buffer (Sigma Aldrich, #A4955, $92.70 for 100ml)
http://www.sigmaaldrich.com/catalog/product/sigma/a4955?lang=en®ion=US
Structure (PDB or Homology model)
-- PDB #: 1YWF
Current Inhibitors: I-A09 (https://www.ncbi.nlm.nih.gov/pubmed/20167798)
Expression Information (has it been expressed in bacterial cells): Expressed in e.Coli
Purification Method: immobilized metal affinity chromatography, and size exclusion chromatography (can also use a cobalt charged resin column)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775457/
http://www.biomedcentral.com/1471-2180/13/292
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MAVRELPGAWNFRDVADTATALRPGRLFRSSELSRLDDAGRATLRRLGITDVADLRSSREVARRGPGRVP
DGIDVHLLPFPDLADDDADDSAPHETAFKRLLTNDGSNGESGESSQSINDAATRYMTDEYRQFPTRNGAQ
RALHRVVTLLAAGRPVLTHCFAGKDRTGFVVALVLEAVGLDRDVIVADYLRSNDSVPQLRARISEMIQQR
FDTELAPEVVTFTKARLSDGVLGVRAEYLAAARQTIDETYGSLGGYLRDAGISQATVNRMRGVLLG
*length of your protein in Amino Acids: 276aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
30.1538 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 14440
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
atggcggtgcgcgaactgccgggcgcgtggaactttcgcgatgtggcggataccgcgacc gcgctgcgcccgggccgcctgtttcgcagcagcgaactgagccgcctggatgatgcgggc cgcgcgaccctgcgccgcctgggcattaccgatgtggcggatctgcgcagcagccgcgaa gtggcgcgccgcggcccgggccgcgtgccggatggcattgatgtgcatctgctgccgttt ccggatctggcggatgatgatgcggatgatagcgcgccgcatgaaaccgcgtttaaacgc ctgctgaccaacgatggcagcaacggcgaaagcggcgaaagcagccagagcattaacgat gcggcgacccgctatatgaccgatgaatatcgccagtttccgacccgcaacggcgcgcag cgcgcgctgcatcgcgtggtgaccctgctggcggcgggccgcccggtgctgacccattgc tttgcgggcaaagatcgcaccggctttgtggtggcgctggtgctggaagcggtgggcctg gatcgcgatgtgattgtggcggattatctgcgcagcaacgatagcgtgccgcagctgcgc gcgcgcattagcgaaatgattcagcagcgctttgataccgaactggcgccggaagtggtg acctttaccaaagcgcgcctgagcgatggcgtgctgggcgtgcgcgcggaatatctggcg gcggcgcgccagaccattgatgaaacctatggcagcctgggcggctatctgcgcgatgcg ggcattagccaggcgaccgtgaaccgcatgcgcggcgtgctgctgggc
*GC% Content for gene: 65.821%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**